Allergens reacted with carbodiimides

ABSTRACT

DESENSITIZING OR IMMUNIZING AGENTS ARE PROVIDED FOR TREATING HYDPERSENSITIVE OR ALLERGIC CONDITIONS. MODIFIED ALLERGENIC MATERIAL IS PREPARED COMPRISING AN ALLERGENIC PROTEIN OR GLYCOPROTECTION CROSSLINKED INTERMOLECULARLY OR INTRAMOLECULARLY WITH AN INORGANIC CYANATE. THE MODIFIED MATERIAL HAS REDUCED ALLERGENICITY RELATIVE TO THE UNCROSSLINKED ALLERGEN AND HAS THE ABILITY TO PRODUCE ANTIBODIES HAVING CROSS-SPECIFICITY FOR THE UNCROSSLINKED ALLERGENIC PROTEIN OR GLYCOPROTEIN.

United States Patent US. Cl. 260-112 R 21 Claims ABSTRACT OF THEDISCLOSURE Desensitizing or immunizing agents are provided for treatinghypersensitive or allergic conditions. Modified allergenic material isprepared comprising an allergenic protein or glycoprotein crosslinkedintermolecularly or intramolecularly with an inorganic cyanate. Themodified material has reduced allergenicity relative to theuncrosslinked allergen and has the ability to produce antibodies havingcross-specificity for the uncrosslinked allergenic protein orglycoprotein.

This is a division of application Ser. No. 59,745 filed July 30, 1970.

The present invention relates to desensitizing or immunizing agentsuseful in the treatment of hypersensitive or allergic conditions, and toa method for their preparation.

It is well known that some individuals are allergic or hypersensitive tocertain allergenic materials such as pollens, house dust, cat fur,cereals and a host of other common substances. Such individuals cansuffer acute discomfort as a result of their allergic conditions whichmay manifest themselves in such diseases as asthma, hay fever, eczema,dermatitis and migraine. Consequently work continues to find suitabletreatments which will alleviate the suffering of the allergic patient.

One technique which has been used in the past in the treatment ofallergic conditions is the so-called desensitization therapy. Thepatient undergoing such therapy is administered repeated graduallyincreasing doses of an extract of the particular allergenic material ormaterials to which he is sensitive. At the end of a course of treatmentthe patients natural resistance to the allergen is usually greatlyenhanced, presumably as a result of the build-up of antibodies in hisbody, stimulated by the administered extract.

As will be appreciated, this desensitization thereapy sulfers fromcertain disadvantages, not the least of which lies in the possibilitiesthat a dangerously high dose of allergen may inadvertently beadministered, resulting in a general anaphylactic reaction in thepatient. It has been suggested that this problem would be overcome if itwere possible to modify the allergenic materials in such a way thattheir allergenicity is reduced relative to their desensitizing and/ orimmunizing properties. In other words, if the allergenic material couldbe rendered harmless, or at least less harmful to the sensitive patient,while at the time retaining its ability to stimulate antibodyproduction, one of the main disadvantages of desensitization therapycould be removed.

According to the present invention there is provided a process for thepreparation of a modified allergenic material, which process comprises.the reaction of an allergenic material with a polyaldehyde, apolyketone, a carbodiimide, an epihalohydrin or an inorganic cyanate,with the proviso that when an inorganic cyanate is employed the reactionis carried out under acid conditions.

3,825,525 Patented July 23, 1974 The allergenic starting material whichis modified according to the process of this invention may be obtainedfrom an allergen-containing substance such as pollen by extracting theallergen-containing substance with a suitable solvent, usually aqueous,in a known manner. The allergenic extract obtained in this way consistsprincipally of protein or glycoprotein, usually contaminated with freecarbohydrate. The allergenic extract is then usually purified byremoving some of the contaminants, e.g. by dialysis, precipitation orgel filtration, and the resulting purified allergenic material may thenbe treated according to the process of the invention. A fullerdescription of some of the techniques available can be found in anarticle by J. N. Newell in the Journal of Allergy, Vol. 13, 1942, pages177 to 203, particularly page 187. In another useful extractionprocedure, the allergen-containing material or an aqueous extractthereof is treated with aqueous phenol and the allergenic extract isrecovered from the phenol phase.

The polyaldehyde and polyketone reagents which lie Within the scope ofthis invention include dialdehyde and diketones as well as the higheraldehyde and ketones. Generally of the polyketones and polyaldehydeswhich may be used we prefer to use dialdehydes, e.g. those having from 2to about 24 carbon atoms in the molecule. The dialdehyde may bealiphatic, cycloaliphatic, heterocyclic or aromatic, and may have eithera straight or branched chain structure, e.g. glyoxal, 1,3-propanedial,1,4-butanedial, glutaraldehyde and c m-dialdehydes having from 14 to 24carbon atoms in the molecule. A particularly preferred dialdehyde isglutaraldehyde.

The term carbodiimide as used in the present specification refers tocompounds of formula (I) and salts thereof:

wherein R and R are the same or different and each is an aliphatic,aromatic or heterocyclic radical. The preferred carbodimides for use inthe present invention are watersoluble, e.g.1-cyclohexyl-3-(Z-morpholinoethyl) carbodiimidemetho-p-toluenesulphonate.

As used herein the term epihalohydrin refers to compounds of formula(II):

wherein X is a halogen atom, particularly bromine or chlorine. We findthat good results are obtained with epichlorohydrin.

As examples of inorganic cyanate ion reagents may be mentioned thealkali metal cyanates, particularly potassium cyanate; and the alkalineearth metal cyanates.

As a general rule, allergenic proteins are fairly resistant todenaturation and can usually be heated to relatively high temperatureswithout denaturation taking place. The process of this invention canthus take place over a wide range of temperatures although in practiceit will generally not be necessary or desirable to exceed atemperatureof about C. When dialdehydes are employed it is preferred tooperate at a temperature below 37 C., and in practice we prefer to carryout the reaction at room temperature in each case, i.e. about 20-25 C.When employing dialdehydes, exteme reaction conditions should be avoidedin order to reduce undesirable byproducts such as are produced by selfcondensation of the aldehydes.

In the case of polyaldehydes and polyketones, the pH that which theprocess of this invention should be carried out is not critical. Asuitable pH range is from about 4 to about 8, with a pH of about 5 beingpreferred. Very low pH values should be avoided in order to reducepossible self condensation of the aldehydes.

Similarly in the case of epihalohydrin and carbodiimides, the reactioncan be carried out at any convenient pH, although we prefer to avoidextremes of pH. For the epihalohydrin the reaction proceeds smoothly atalkaline pHs, suitably at about pH 8. In the case of the carbodiimide,the reaction is preferably carried out at acid pHs, suitably at pH about5.

The reaction of the allergenic protein with an inorganic cyanate reagentin the process of this invention must be carried out at acid pHspreferably at a pH of about 5.

The reagents used in the process of this invention are believed to actas cross-linking agents for the allergenic material, forming interand/orintra-molecular links. Thus, although the allergenic starting materialsare watersoluble, the modified allergenic products of the presentinvention are usually only sparingly soluble. The solubility of themodified allergenic materials appears to be dependent on the extent ofcross-linking, which in turn may vary according to such factors as thenature and quantity of reagent used and the reaction conditions. We arenot aware of any empirical method of determining in advance thesolubility characteristics of the modified allergenic products andconsequently the reaction variables necessary to obtain a product havingthe desired solubility must be determined by trial and error. This is amatter of routine, however, and should present little difiiculty tothose skilled in the art.

In another embodiment of this invention there is provided apharmaceutical preparation comprising the modified allergenic materialprepared by the process described above, and a parenterally acceptablecarrier. Suitable carriers include isotonic salt or buffer solutions,oily carriers and other materials well known in the art. If desired anadjuvant such as tyrosine, alumina, aluminium hydroxide or aluminiumphosphate, may be included.

The modified allergenic material is generally administered bysubcutaneous injection. Dosage rates will vary according to the allergiccondition of the patient.

Generally, in preparing pharmaceutical compositions in accordance withthis invention, the modified allergenic material should be one which hasa reduced solubility relative to the starting material, but which issufficiently soluble in vivo to provide the desired releasecharacteristics. We find that one way of achieving this is to use asparingly soluble modified allergen absorbed on alumina and likematerials. The degree of solubility of the crosslinked allergenicmaterial is modified by absorption onto the alumina and the optimum invivo release characteristics can be achieved by routine experimentation.

On parenteral administration to animals of the modified allergenicmaterials of this invention, it has been found that appreciable levelsof circulating antibody are formed, the antibody havingcross-specificity for the unmodified allergenic material. The modifiedmaterials tested did not cause any anaphylactic reaction inhypersensitive animals.

EXAMPLE 1 Preparation of Insoluble Allergen Derivative WithGlutaraldehyde A partially purified extract of Cocksfoot pollen,containing 10 mg. protein/ml. in 0.1M sodium acetate buffer pH 5.3 (5ml.), was treated with 0.5% glutaraldehyde solution (5 ml.), and stirredat room temperature for 30 minutes. The precipitate which formed wasremoved by centrifugation and washed with distilled water to removeresidual soluble material. It was finally suspended in phenol-salinesolution for storage.

Test of Allergenicity The suspension of insolubilized pollen at aconcentration of mg./ml. was pricked into the skin of grasssensitiveallergic patients. At the same time the starting material andphenol-saline solution respectively were pricked into other areas ofskin of the same patients. Weal areas were measured after 10 minutes,and are expressed in Table I in sq. mm. It can be seen thatinsolubilized material at 200 times the concentration of startingmaterial has barely one tenth of its allergenicity: the retainedallergenicity is thus negligible when compared with that of the startingmaterial.

Test of Immunogenicity The insolubilized material and the startingmaterial respectively were emulsified in Freunds complete adjuvant togive concentrations of 1 mg./ml. Groups of guineapigs were injectedsubcutaneously with one or other of the emulsions (0.5 ml.). After 22days the animals were clipped free of hair on the flanks and a series ofintradermal injections (0.1 ml.) of a serial dilution of purifiedCocksfoot pollen extract was made into the clipped areas. A 5% solutionof Pontamine Sky Blue (0.4 ml.) was im mediately injected intravenously.After 20 minutes Weal diameters were measured, and are recorded in TableII.

It can be seen that not only does the glutaraldehydetreated materialproduce antibody which reacts with the starting material, but it is amore eflicient immunizer than the starting material.

TABLE 1 Insolubilized cocksfoot Purified pollen cocksfoot extract,extract, Phenol- Patlent number 10 mg.lml. 50 pgJm]. saline Total 65 2244 Total less that for phenolsaline. 17 176 TABLE II Weal diameter (mm.)for quantity of cocksfoot pollen extract injected intradermally Guineapig Immumzing material 100 10 1 0. 1 0. 01 0. 001 number; (0.5 mg.) g.g. g. ug. ng. g

1 15 0 0 0 0 2 Purified cocksfoot 16 0 O 0 0 g 3. pollen extract. 17 0 00 0 0 4 16 0 0 0 0 0 5 14 0 0 0 0 0 6 12 10 9 0 0 0 7- Glutaraldehyde-14 13 12 12 10 8 8 treated cocksfoot 16 14 12 11 10 8 9. pollen extract.12 10 0 0 0 0 10 12 9 0 0 0 0 EXAMPLE 2 Preparation ofCarbodiimide-Treated Allergen Fifty mg. of material which had beenextracted from Test of Allergenicity A suspension of the test materialand a solution of the starting material, both in phenol-saline, and themedium itself, were respectively pricked into the skin of grass vsensitive allergic 'patients. Weal areas were measured after minutes.

The results of the allergenicity test are shown in Table III:

6 grass sensitive allergic patientsLWeal areas were measured after 10minutes. The results of the test of allergenicity are shown in Table V:

5 TABLE III TABLE V Carbodi- Epichlor- Mixed imidehydringrass treatedMixed treated pollen mixed grass mixed grass extract, grass pollenPhenol 1Q pollen pollen ex- Patient number 50 g./m1. extract salineextract. 50 tract, 1 mg./ Phenol Patient number g./m m saline 12 0 0 3 20 62 0 0 8 0 0 1 0 12 0 0 12 0 0 15 0 0 8 2 0 62 3 0 3 0 0 12 0 0 Total112 5 0 Note.Figu.res indicate weal areas (sq. mm.).

Test of Immunogenicity Replicate guinea pigs were immunized bysubcutaneous injection of an emulsion containing the test material inFreunds complete adjuvant. A similar set of guinea pigs were immunizedwith the unmodified starting material adjuvanted in the same way. After21-8 days the animals were clipped free of hair on the flanks and aseries of intradermal injections (0.1 ml.) of a serial dilution ofstartnig material terminating with normal saline was made into theclipped area. I

A 5% solution of Pontarnine Sky Blue (0.4 ml.) was immediately injectedintravenously. After minutes bluecolored weals formed at some of thesites of intradermal injection, indicating the presence of antibody withspecificity for the starting material. Weal Diameters were measured andrecorded.

The results of the immunogenicity tests are recorded in Table IV:

TABLE IV Weal diameter (mms.) for quantity of mixed grass pollen extractinjected intradermally Guinea pig immunizing material, 110 10 1 0.1 0.010. 001 number 5 mg. g. g. g. g. g. g.

14 12 10 9 8 8 Mixed grass pollen 14 13 10 9 8 extract. 16 14 12 10 9 914 13 10 Carboddiimide-treiaited 0 l0 9 mlXB grass p0 en extract. {a 3 ii It can be seen from the results shown in Tables III and IV that higherlevels of antibody were produced by immunization with the carbodiimidemodified allergen, but that the allergenicity of the material wasnegligible.

EXAMPLE 3 Test of Allergenicity A suspension'of the test material and asolution of the starting material, both in phenol-saline, and the mediumitself, were respectively pricked into the skin of NorE.-Figuresindicate weal areas (sq. mm.).

Test of Immunogenicity Replicate guinea pigs were immunized bysubcutaneous injection of an emulsion containing the test material inFreunds complete adjuvant. A similar set of guinea pigs were immunizedwith the unmodified starting material adjuvanted in the same way. After21-8 days the animals were clipped free of hair on the flanks and aseries of intradermal injections (0.1 ml.) of a serial dilution ofstarting material terminating with normal saline was made into theclipped area.

A 5% solution of Pontamine Blue Sky (0.4 ml.) was immediately injectedintravenously. After 20 minutes bluecolored weals formed at some of thesites of intradermal injection, indicating the presence of antibody withspecificity for the starting material. Weal diameters were measured andrecorded. The results of the test of immunogenicity are shown in TableVI:

: Mixed grass pollen extract (0.5 mg.).

Epichlorhydrintreated grass pollen extract (0.5 mg.).

It can be seen from the above results that detectable levels of antibodywere produced by immunization with epichlorhydrin-treated allergenswhile the allergenicity was reduced to a negligible level.

EXAMPLE 4 50 mg. of material which had been extracted from Cocksfootgrass pollen was dissolved in 10 ml. of 0.01M ph 8.0. 1 ml. of1-br0mo-2z3 epoxypropane was added slowly and the mixture was stirredovernight at room temperature. The precipitate which formedwas-centrifuged off, washed with phosphate buffer, and suspended inphenol saline. I

Results of the biological testing are shown in Tables III and IVJThetesting procedures were identical with those of Example 3'. Detectablelevels of antibody were produced by immunization, while allergenicitywas reduced to negligible levels.

TABLE VII.-IMMUNOGENICITY OF EPIBBOMOHYDRIN- TREATED ALLERGEN Wealdiameter (mm.) [or quantity of cockstoot; pollen extract injected i.d.Guinea g Immunizing 100 10 1 0. 1 01 0. 001 number material g. g. g. g.g. g.

1- Cocksfoot pollen 16 extract 16 3 (0.5 mg.). 16 4 Epibromohydrln 14 5treated cocksfoot 14 6 extract (0.6 mg.).

TABLE VIIL-ALLEBGENICITY 0F EPIBROMOHYD RIN- TREATED ALLERGENEpibromohydrintreated cocksioot Cocksioot pollen exextract, tract; 100Phenol Patient number 100 g./ml. g./ml. saline Total 70 0 0 EXAMPLE 5Preparation of Cyanate-Modified Allergen An aqueous extract of mixedgrass pollens (Bent, Broom, Cocksfoot, Dogstail, False Cat, Fescue,Foxtail, Meadow Rye, Timothy Vernal and Yorkshire Fog) was partiallypurified by treatment with aqueous phenol followed by precipitation ofprotein from the phenol phase to remove much of the carbohydrate and thelow molecular weight materials. 50 mg. of the resultant partiallypurified allergenic protein was dissolved in 5 ml. of water and the pHadjusted to pH 8 by the addition of sodium hydroxide solution. The pHwas then further adjusted 5.0 with acetic acid, 0.2 g. of potassiumcyanate was added, and the mixture stirred at room temperatureovernight. The precipitate which formed was washed repeatedly withphosphate buffer at pH 8 and then suspended in phenol saline.

Test of Allergenicity A suspension of the cyanate-modified allergenprepared as above, a suspension of the unmodified partially purifiedallergen extract (both in phenol saline) and the phenolsaline mediumitself were respectively pricked into the skin of grass-sensitiveallergic patients. Weal areas were measured after ten minutes and areexpressed in Table XVII in sq. mm. It can be seen that the cyanatemodified allergen exhibits negligible allergenicity relative to theunmodified material.

Test of Immunogenicity Replicate guinea pigs were immunized bysubcutaneous injection of an emulsion containing the cyanate-modifiedallergen in Freunds complete adjuvant. A similar set of guinea pigs wereimmunized with the unmodified starting 8 material adjuvanted in the same,way. After 21-28 days both sets of animals were clipped free of hair onthe flanks and a series of intradermal injections (0.1 ml.) of a serialsolution of the starting material terminating with normal saline wasmade into the clipped area.

A 5% solution of Pontamine skyblue (0.4 ml.) was immediately injectedintravenously. After minutes blue-colored weals formed and some of thesites of intradermal injection, indicating the presence of antibody withspecificity for the starting material. Again weal diameters weremeasured and are recorded in Table X. It can be seen that the cyanatemodified allergen retains the immunizing specificity of the startingmaterial.

TABLE X Weal diameter (mms.) for quantity of mixed grass pollen extractinjected Ld. Guinea g 100 10 1 0. 1 0. 01' 0. 001 number Immunizingmaterial g. g. g. g. g. g

14 Mixed grass pollen 14 extract (6 mg.). 16 14 12 cyanate-treated 12mixed grass pollen 10 2 5 extract (5 mg.) Mixed grass pollen 12 11--extract (0.5 mg.). 14 12 2 EXAMPLE 6 In order to obtain confirmation ofthe biological testing results the procedure of Example 5 was repeated.The results of the test of the allergenicity of the modified allergenare recorded in Table XI and the results of the tests of theimmunogenicity of the cyanate-modified allergen are recorded in TableXII.

TABLE XI Cyanate- Mlxed treated grass mixed pollen grass extract, pollen100 extract, 2 Phenol Patient number g./ml. mg./m1. saline Total 60 1 1Norm-Figures indicate weal areas (sq. mm.).

TABLE XII Weal diameter (ms) for quantity of mixed grass pollen extractinjected i.d.

Guinea pig 100 10 1 0.1 0.01 0.001 number Immunizing material g. g. pg.g. pg. g.

Mixed grass pollen extract (5 mg.).

mixed grass pollen extract (5 mg.).

9 10 Mixed grass pollen extract (0.5 mg.).

Cyanate-treated mixed grass pollen Cyanate-treated extract (0.5 mg.).

EXAMPLE 7 Treatment of Pollen Extract With Phenyl Glyoxal Method: A 3%solution of phenylglyoxal hydrate dissolved in 0.01M phosphate butter,pH 8, was added to an equal volume of 50 mg. of material which had beenextracted from Cocksfoot pollen and which, after purification bytreatment with phenol, was dissolved in the same butter. The recationwas carried out at room temperature for 24 hours. The precipitate whichformed was washed repeatedly with phosphate buffer at pH 8 and thensuspended in phenol saline Test of Allergenicity: A suspension of thephenylglyoxal-modified material prepared as above, a suspension of theunmodified phenol treated Cocksfoot extract (both in phenol-saline) andthe phenol-saline medium itself were respectively pricked into the'skinof grass-sensitive allergic patients. Weal areas were measured after tenminutes and are expressed in Table XIII in sq. mm.

) Test of immunogenicity: Replicate guinea pigs were immunized bysubcutaneous injection of an emulsion containing the phenylglyoxalmodified material in Freunds complete adjuvant. A similar set of guineapigs were immunized withihe unmodified starting material adjuvanted inthe same way. After 28 days, both sets of animals were clipped free ofhair on the flanks and a series of intra- 1 0 were respectively prickedinto the skin of grass-sensitive allergic patients. Weal areas weremeasured after 10 minutes and are expressed in Table XV in sq. mrn.

TABLE XV Alumina adsorbed glutar- Alumina aldehyde adsorbed Timothytreated untreated pollen Timothy Timothy aqueous pollen pollen extract,extract, extract, Phenol Patient number 50 ig/ml. 1 mg./ml. 1 mg.lm1.saline Test of immunizing specificity: Replicate guinea pigs wereimmunized bysubcutaneous injection of the alumina-glutaraldehydeadsorbed modified material. A similar set of guinea pigs were immunizedwith unmodified alumina-adsorbed timothy pollen extract at a pollenconcentration of 1 mg./ml. 21 days later a repeat injection was givenand the animals bled 10 days later.

Passive cutaneous anaphylaxis was carried out on the sera by injectionof ser ial dilutions intradermally into a clipped area on the flanks ofa set of guinea pigs. The pigs were then challenged intravenously withthe unmodified aqueous timothy extract, containing 0.4 ml. Pout-aminesky blue solution, five hours later. Results obtained with differentconcentrations of challenging antigen are shown in Table XVIII for themodified material and Table XVH for the control suspension withoutglutaraldehyde.

dermal injections (0.1 ml.) of a serial solution of the TABLE XVIstarting material terminating with normal saline was made Challenge intothe clipped area. A 5% solution of Pontamine sky $32? blue (0.4 1111.)was immediately injected intravenously. aqueous Weal diameters in mm. atserum dilution After 20 minutes blue colored weals formed at some of ggg the sites of intradermal injection, indicating the presence Guinea pigextract, of antibody with specificity for the starting material. numbermgJml' 1/5 1/25 1/125 1/625 1/3125 5811M Weal areas were measured andare recorded in Table 1 XIV 10 TABLE XIV W r r eal diameter mm. orquantit o V Cocksioot pollen extractiniected .d. TABLE XVII Guinea pig 110 1 0.1 0.01 0.001 Challenge number Immunlzing material g. pg. g. ag.g. g. :3 aqueous iIII Cockfoot pollen 14 14 i it 7 Timothy Wealdiameters in mm. at serum dilution 3. tract (5 mg.). 10 7 pollen of- 9 8ii pig irii 1 5 25 1 625 1 3125 s 11 I! Phenylglyoxal treated P m er mg//1 1/ a ne cockstoot pollen ex- 1 25 16 41:11:: (0511159- 100 20 1sEXAMPLE 8 Treatment of Pollen Extract with Glutaraldehyde and SubsequentAbsorption Into Aluminium Hydroxide Method: 1 ml. of 5% glutaraldehydesolution was added to 9 ml. of an aqueous extract of timothy pollencontaining 1 mg./ml. at pH 5.3. The mixture was stirred at roomtemperature for 3 days. 6 ml. of 2% aluminium hydroxide solution wereadded and the mixture stirred for one hour. After centrifugation theprecipitate was washed twice in 0.01M phosphate buffer pH 8 and threetimes in distilled water and finally resuspended to a concentration of 1mg./ml. pollen in phenol saline. The procedure was repeated without theaddition of glutaraldehyde, the final suspension serving as a control inthe immunizing specificity test.

Test of allergenicity: A suspension of the above material, a solution ofthe unmodified pollen extract (both in phenol-saline) and the phenolsaline medium itself It can be seen that treatment with glutaraldehydeleads to enhanced antibody formation when compared with that fromuntreated material, although allergenic activity was reduced.

We claim:

1. A process for the preparation of a modified allergenic material frompollen and house dust which comprises reacting an allergenic extract ofpollen or house dust which consists principally of a protein orglycoprotein with a canbodiimide at an acidic pH to reduce the'allergenicity and recovering the modified allergenic material soproduced.

2. A process according .to claim 1 wherein the temperature is below C.

3. A process according to claim 1 wherein the pH is about 5.

4. A process according to claim 2 wherein the temperature is roomtemperature.

-1 l 5. A process according to claim 1' wherein the carbodiimide is ofthe formula or a salt thereof wherein R and R are the same or differentand are each an aliphatic, aromatic or heterocyclic moiety.

6. A process according to claim 1 wherein the carbodiimide iswater-soluble.

7. A process according to claim 1 wherein the carbodiimide is1-cyclohexyl-'3 (2-morpholinoethyl) carbodiimidemetho-p-toluenesulphonate.

8. A process according to claim 1 wherein the allergenic extract is apollen extract.

9. A process according to claim 1 wherein .the allergenic extract is ahouse dust extract.

10. A process according to claim 8 wherein the pollen is Cocksfootpollen.

11. A process according to claim 8 wherein the pollen is mixed grasspollens.

12. A process according to claim 8 wherein the pollen is timothy pollen.

13. A modified allergenic material which comprises an allergenic extractof pollen or house dust which consists principally of a protein orglycoprotein, which allergenic extract is crosslinked interorintra-molecularly with a carbodiimide, said modified allergenic materialbeing substantially water-insoluble or only sparingly water-soluble andhaving a reduced allergenicity relative to the uncrosslinked protein orglycoprotein and having the ability to stimulate the production ofantibodies having crossspecificity for the unlinked protein orglycoprotein.

' 14. A modified allergenic material according to-claim 13 wherein theallergenic extract'is'a -pollen -extract -.l

15. A modified allergenic material according to claim '13 wherein theallergenic extract isa house dust-extract. 16. A modified allergenicmaterial: according toclaim 13 wherein the carbodiimide is of theformula I R- -N,=C=NR' i or a salt thereof whereinR and R' are thesameor difierentand areeac'h an aliphatic, aromatic or heterocyclicmoiety; t 17. A modified allergenic material accordin g to claim 13wherein the carbodiimide is water-insoluble.

18. A modified allergenic material according to claim 13 wherein thecarbodiimide is 1cyclohexyl-3- (2-morpholinoethyl) carbodiimidemetho-p-toluenesnlphonate.

19. A modified allergenic material according to claim 14 wherein thepollen is Cock-sfoot pollen.

20. A modified allergenic material according to claim 14 wherein thepollen is mixed grass pollens; f 21. A modified allergenic materialaccording to claim 14 wherein the pollen is timothy pollen. j

References Cited UNITED STATES PATENTS 2,938,892 5/1950 Sheehan =250 i 2OTHER REFERENCES I Chem. Abstracts, Vol. 73, "1970, Overell 7260a.

HOWARD E. SCHAIN, Primary Examiner C "UNITED STATES PATENT OFFICECERTIFICATE OF CORRECTION menu, 3,825,525 Dated July 23,1974

Inventor) Noel Austin Mullen et a1.

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected asshown below:

IN THE ABSTRAC'L OF THEY DISCLOSURE Colunni 1., 1ine20, cflan ge "eninorganic cyanate" to a carbodiimide Signed and sealed this 26th day ofNovember 1974.

(SEAL) Attest:

mcc'o'iz' MI" 'clssoiffim c. MARSHALL 1mm 7 Commissioner of PatentsAttesting Officer FORM PC4050 (10-69)

